Abstract

Centriole-to-basal body conversion, a complex process essential for ciliogenesis, involves the progressive addition of specific proteins to centrioles. CHIBBY (CBY) is a coiled-coil domain protein first described as interacting with β-catenin and involved in Wg-Int (WNT) signaling. We found that, in Drosophila melanogaster, CBY was exclusively expressed in cells that require functional basal bodies, i.e., sensory neurons and male germ cells. CBY was associated with the basal body transition zone (TZ) in these two cell types. Inactivation of cby led to defects in sensory transduction and in spermatogenesis. Loss of CBY resulted in altered ciliary trafficking into neuronal cilia, irregular deposition of proteins on spermatocyte basal bodies, and, consequently, distorted axonemal assembly. Importantly, cby(1/1) flies did not show Wingless signaling defects. Hence, CBY is essential for normal basal body structure and function in Drosophila, potentially through effects on the TZ. The function of CBY in WNT signaling in vertebrates has either been acquired during vertebrate evolution or lost in Drosophila.

CBY conservation in animals. (A) CBY is a small protein with a conserved coiled-coil domain close to the C terminus, highly conserved in animals (black bar). Each of the eight classes of amino acids is represented by one color. (B) Orthologs of CBY are found in animals with motile cilia (black filled circles) and in the closest unicellular relatives of animal, the marine choanoflagellate Monosiga brevicollis, but not in nematode genomes or in most unicellular organisms (open circles). A probable homolog is found in the parabasalid protist Trichomonas, and a more distant relative is found in the club moss S. moellendorffii (gray circle). CBY is not found in most bikonts (open circles).

cby is expressed in ciliated neurons of the Drosophila PNS. (A) Transgenic flies expressing cby coding sequences fused to GFP under cby regulatory sequences were analyzed for GFP expression. GFP expression is detected only in type I ciliated cells of the PNS in Rfx+/+ flies (left). GFP staining is observed in the cytosol and mainly as a dot at the tip of the dendrite. The PNS is labeled (red) with an anti-Futsch antibody (22C10). In Rfx−/− flies, the cby reporter construct is not expressed (right). (B) Double labeling of a transgenic fly strain expressing cby coding sequences fused to the fluorescent reporters GFP or Tomato under cby regulatory sequences with different markers of the centriole. CBY is localized at the tip of the centriole/basal body, as observed with ASL, PLP, SAS4, UNC fused to GFP, and CG14870 fused to a MYC epitope. CBY colocalizes with the TZ marker CG14870.

cby is expressed during spermatogenesis. (A) The diagram shows the cycle of the centriole during spermatogenesis. In G2 spermatocytes, the two pairs of replicated centrioles elongate and reach the membrane before meiosis entry, and single centrioles are distributed to each of the haploid spermatids after meiosis. (A1–3) CBY-GFP is localized at the tip of the elongating centrioles labeled for γ-tubulin (γTUB) from early G2 spermatocytes to early spermatids. (A4 and 5) As sperm axoneme elongates, CBY is found at the tip of the axoneme and lost from the basal body (white arrows). Note that some CBY-GFP is found associated with mitochondria during spermatid elongation (white arrowhead). (A6–10) CBY-TOMATO localization in live squashes of Drosophila testis expressing UNC-GFP. (A6–8) CBY is localized at the tip of the elongating centriole adjacent to the UNC-GFP staining from spermatocytes to early spermatid stage. CBY-TOMATO is transiently associated with the mitochondria (arrowhead). (A9 and 10) In elongating spermatids, CBY-TOMATO is associated with UNC-GFP only at the tip of the axoneme (A9) and is no longer present with UNC-GFP at the basal body apposed to the nuclei at the spermatid heads (A10). (A4–10) Nuclei are labeled with Hoechst. (A1 and 6) Early G2 spermatocytes. (A2 and 7) Late G2 spermatocytes. (A3 and 8) Round spermatids. (A4, 5, 9, and 10) Elongating spermatids. (B) CBY-TOMATO expression is similar in Rfx+/+ and Rfx−/− spermatocytes and spermatids. Centrioles are labeled with γ-tubulin antibody. Bars, 5 µm.

Inactivation of cby leads to defective behavior and mechanosensation. (A) Diagram of the cby genomic locus. The second coding exon was replaced by homologous recombination with a miniwhite gene flanked by two LoxP sites. The primers designed to screen for the recombinant events are indicated in yellow for the left arm and red for the right arm. (B) Diagram representing the bang assay to evaluate fly coordination. The number of flies that reach a defined level in the tube is counted 1 min after tapping the tube. cby1/1 flies and cby1/Df(3R)BSC805 (cby1/Df) stay at the bottom of the tube compared with control flies. The phenotype is rescued by adding one copy of the cby-Tomato transgene. n = 30 flies per assay. Error bars represent the SD of five assays. (C) Antennal nerve potentials recorded from age-matched sib cby/TM6C heterozygotes (cby/+), cby homozygotes (cby/cby), and from Df(3R)BSC805/TM6B (Df/TM6B) and cby/Df(3R)BSC805 (cby/Df) sibs in response to a five-pulse sound stimulus (stim). Each trace represents the averaged responses to 10 stimuli. Traces shown are those closest to the mean values for their respective genotypes. The third trace is representative of mutant recordings that do not show discernible SEP peaks. (D) Summary of sound-evoked nerve potential amplitudes. Mean and median peak amplitudes of the antennal SEPs for each indicated genotype. Mean values are derived only from those recordings showing discernible peaks; medians are from all recordings.

Ultrastructural defects of the chordotonal cilia in cby-deficient flies. (top left) Scheme and longitudinal section of a typical leg or antennal CO composed of two neurons ensheathed by a scolopale cell and linked to the cap cell at the tip of the ciliary endings. The cilia are anchored on a distal basal body (DBB) aligned above the proximal basal body (PBB) at the end of the dendrite. (a1–5) Sections at the level of the ciliary axoneme. In control legs (a1), axonemes are composed of nine peripheral microtubule doublets. (a2–5) cby1/1 axonemes show a reduced number of microtubule doublets (a2 and 3), altered symmetry (a4), or extra microtubules (a5). (b1–3) Sections at the distal TZ. (b1) In control samples, a dense ring structure of ninefold symmetry is observed. (b2 and 3) In cby1/1 antennae, interrupted, incomplete, and misformed ring structures are observed. (c1–3) Sections at the proximal TZ level. (c1) In control TZ, decorations connecting the membrane can be seen and likely correspond the Y links. (c2 and 3) In cby1/1 antennae, decoration are interrupted (black arrows; c2), or the ninefold symmetry of the TZ decorations is interrupted (c3). (d1–3) Sections at the level of the proximal basal body. Bars, 0.5 µm. (d1) Basal bodies are anchored to the cell membrane by dense fibers in control flies (black arrow). Tight junctions connect the two dendrites. (d2 and 3) Two examples showing missing basal bodies in cby1/1 flies. (e1–3) Longitudinal sections of cby1/1 ciliary endings. In several cases (e1 and 2), the TZ is discontinuous (white arrows). (e2 and 3) Membrane bulges are observed along the cilia (black arrows). (f1–3) Low magnifications of transverse sections of scolopidia. Bars, 0.5 µm. (f1) Two axonemes are present in control antennae. (f2 and 3) Only one or no axoneme is sometimes observed in cby1/1 antennae.

CBY is required for IFT protein distribution. (A–D) Expression of NompB/IFT88 (A and B) and CG11356/ARL13b (C and D) in either control (A and C) or cby1/1 embryonic chordotonal cilia (B and D). (A and B) NompB-GFP accumulation is increased in cby1/1 cilia compared with control cilia (white arrow). (C and D) CG11356-GFP accumulates continuously in control cilia, but its accumulation is punctuated in cby1/1 cilia (white arrow). The overall abundance of CG11356 is also reduced in mutant cilia.

CBY is required for axonemal organization in the testis. (A) cby1/1 mutant testes still produce mature spermatozoids, but their number is reduced. The spermatid cysts are disorganized, and the number of spermatid per cysts is often compromised. (B) Young spermatid cysts are disorganized and contain a reduced number of spermatids. Some axonemes are not associated with mitochondrial derivatives (white arrowhead), and some mitochondrial derivatives are not associated with an axoneme (white arrow). (C) In addition, a few axonemes are misorganized in mutant flies (white arrows) with mainly missing doublets. Bars, 0.5 µm. (D) High magnification of young spermatids highlighting the ultrastructural defects that can be observed on growing axonemes (white arrow). Bars, 200 nm.

UNC distribution associated with basal body function is altered in cby1/1 testis. (A–H) Distribution of UNC-GFP and γ-tubulin (γ-TUB) was observed by antibody staining in fixed testis from control (A and C) or cby1/1 (B and D) flies or by GFP fluorescence in live squashed preparations of control (E and G) or cby1/1 testis (F and H). (A and B) The extent of UNC protein deposition is irregular in cby1/1 spermatocytes compared with control. The two apposed centrioles stained for γ-tubulin show equivalent UNC labeling in control but not in cby1/1 spermatocytes (white arrow). (C–F) UNC labeling marks an increased length segment in cby1/1 early spermatids (white arrows). (G and H) In cby1/1 elongating spermatids, UNC staining at the basal body is diffuse and not limited to a dot, as shown in controls. Some faint UNC staining, not observed in controls, is also observed along the axoneme. The arrow points to the reduced and diffuse UNC staining at the base of the nucleus in cby mutant testis. (E–H) DNA stained with DAPI.